This application is a continuation-in-part of copending U.S. serial no. 08/006,478, filed January 20, 1993.

This invention relates to retroviral vectors that express multiple polypeptide subunits of a eukaryotic protein from a single polycistronic mRNA and the proteins produced from these vectors. The expressed proteins are particularly useful for inducing transplantation tolerance.

Retroviral vectors are useful as agents to mediate gene transfer into eukaryotic cells. These vectors are generally constructed such that the majority of sequences coding for the structural genes of the virus are deleted and replaced by the gene(s) of interest. The retroviral vector contains the signals required for packaging of the virus but does not contain the information required for the generation of an infectious virus particle. The retroviral vector (in the form of a plas id DNA) is transfected into a packaging cell line, which is capable of supplying, in trans, the retroviral gene product(s) necessary to generate a virus particle. The packaging cell line is incapable of giving rise to a retrovirus in the absence of the retroviral vector. Virus produced by the transfected packaging cell line may

) be used to infect other cells, including retroviral packaging cell

lines and hematopoietic cells. The retroviral vector sequences m be integrated into the host genome via retroviral mediat integration and the genes of interest contained within t retroviral vector may be stably expressed off the integrat proviral form. It is necessary that the packaging cell lines a generated in such a fashion as to minimize the potential f recombination between the retroviral structural genes and t sequences present in the retroviral vector. If such recombinati were to occur infectious retroviral particles might be generat which could replicate in any host cell.

The genes of interest may be incorporated into the provir backbone in several general ways. The most straightforwa constructs are ones in which the structural genes of the retrovir are replaced by a single gene which is then transcribed under t control of the viral regulatory sequences within the long termin repeat (LTR) . Other vectors contain promoters in addition to t viral promoter contained in the 5' LTR.

Recently, retroviral vectors have been designed to include t use of internal promoter elements to initiate transcription. O potential problem with retroviral vectors containing multip transcription units is that, if selection is applied for one gen expression of the other gene can be reduced or lost completel This has been termed "promoter suppression" (Emerman & Temin, 198 Nuc. Acids Res., 14: 9381-9396 ). This phenomenon of promot suppression is the reason that the most recent literature repor in the field have concluded that multiple gene expression from single transcript is preferred (Adam, M. A. et al., 1991, J. Viro 65: 4985-4990; Ghattas, I.R. et al., 1991, Mol. Cell. Biol. 1 5848-5859 and Morgan, R.A. et al., 1992, Nuc. Acids. Re 20: 1293-1299 ).

Gene expression of multiple genes from a single eukaryot

transcript was first observed in the picorna viral mRNAs. It ha been well documented that the 5' untranslated sequence of thes viral RNAs contains a sequence which acts as an internal ribosom entry site (IRES) and which functions to facilitate protei translation from sequences located downstream from the first AUG o the mRNA. Using a picorna viral IRES sequence three genes hav been expressed from a single construct, in which one of the gene is transcriptionally driven by the SV40 promoter and the remainin two by the retroviral LTR promoter (Adam et al., supra). In on instance two IRES sequences were used in one single transcrip (Morgan, et al., supra.).

Macejak and Sarnow, ((1991) Nature, 353: 90-94) reported tha the 5' untranslated sequence of the immunoglobulin heavy chai binding protein (BiP, also known as GRP 78, the glucose-regulate protein of molecular weight 78,000) mRNA can directly confe internal ribosome binding to an mRNA in mammalian cells, in a 5' cap independent manner, indicating that translation initiation b an internal ribosome binding mechanism is used by this cellula mRNA.

This invention represents the first use of the BiP IRE sequence in a retroviral vector. It also represents the first us of more than two IRESs for translation initiation from a singl retroviral transcript, as well as the first use of multiple copie of the same IRES in a single construct. Further, it provides th potential, for the first time, to independently express 4 to polypeptides from a single retroviral construct. The inventio further makes possible the production of multiple polypeptides b a single retroviral construct that are capable of forming a single functional, multimeric protein. A particularly valuable embodimen of this invention provides the first instance of this type o retroviral approach to express heterodimeric majo histocompatibility proteins on the cell surface. In particular, th

vectors may be used to express xenogeneic proteins for the specifi purpose of inducing transplantation tolerance in potentia transplant recipients.

The retroviral vectors of the invention have been constructe to have several 5' untranslated regions (UTR) derived from eithe eukaryotic cellular DNA or viral sequences that act as interna ribosome binding sites for multiple translation initiations from single eukaryotic transcript. The 5' untranslated region preferred in accordance with the invention may be multiple copie of a 210 base pair fragment corresponding to the 5' untranslate region of human immunoglobulin heavy-chain binding protein (BiP mRNA. Alternatively, the vectors may include IRES sequences tha are derived from picorna viral sequences as well as the BiP IRE sequence. The vectors are capable of producing severa polypeptides that, when posttranslationally linked form, functiona heteromultimeric proteins. The proteins produced with the vector of the invention are particularly those useful for inducin transplantation tolerance, including several different majo histocompatibility complex (MHC) heterodimeric proteins. Th invention also provides a process for constructing these retrovira vectors.

Abbreviations are: LTR - retroviral long terminal repeat; BiP -IRE sequence from the human immunoglobulin chain binding protein; GPT sequence of the E. coli gpt gene that encodes the enzyme xanthine guanine phosphoribosyl transferase; CAT -sequence of the E. col gene that encodes the enzyme chloramphenicol acetyl transferase Neo - sequence of E.coli Tn5 gene that encodes the enzyme neomyci phosphotransferase; DRα and DRjS - mini-swine MHC II DRα and DR (c haplotype) cDNA genes; EMC - the IRES sequence fro encephalomyocarditis virus. Transfected GP/E-envAM12 cells ar distinguished by the prefix AM, while transfected GP/E-86 cells ar distinguished by the prefix GP. Transduced PA317 cells with viru

generated from GP/E-86 cells are identified by the prefix GPA.

Figure 1 graphically illustrates the retroviral vector pL7gCAT an each of vectors pPBMl through pPBM5, and pPBM7 through pPBMlO, eac of which is briefly described as follows:

pL7gCAT: This retroviral vector is a derivative of pLNCX(Mille and Rosman, 1989, Biotechniques, 7: 980-990) and is capable o expressing three different genes under the translational control o three BiP IRES. This vector also contains two reporter genes whic encode chloramphenicol acetyl transferase (CAT) and xanthine guanine phosphoribosyl transferase (GPT) .

pPBMl: The 3'LTR of M-MLV present in pL7gCAT has been replace by the 3'LTR of M-MPSV (murine myeloproliferative sarcoma virus).

pPBM2: In order to achieve detectable transient expression o the genes from COSM6 cells, a copy of the enhancerless SV40 origi of replication was inserted before the 3'LTR at the Clal site, thereby generating pPBM2. A unique Bglll site was created durin the construction of pPBM2. pPBM2 is designed to express GPT an CAT.

pPBM3: A copy of the mini-swine MHC class II DR_ cDNA gen (Gustafsson et al. 1990, J. Immunology, 145: 1946-1951) wa inserted in pPBM2 after the first BiP IRES, between the HindiII an NotI sites. pPBM3 is designed to express DRjS , GPT and CAT.

pPBM4: A polylinker sequence was inserted at the EcoRI site i pPBM3. This modification potentiates the introduction of anothe gene which would be translated from the 5'LTR. The EcoRI site wa destroyed during this modification. pPBM4 is designed to expres DR0 , GPT and CAT.

pPBM4-prime: The orientation of the SV40 ori fragment w reversed from its orientation in pPBM4. During the construction o pPBM4-prime the polylinker sequence was eliminated from the vecto the EcoRI site was re-created and one of the Clal sites w destroyed.

pPBM5: The neomycin resistance gene was inserted downstream the second copy of the BiP IRES sequence, between the NotI a Bglll sites of pPBM4. pPBM5 is designed to express DR/3 and t neomycin resistance gene.

pPBM7: The mini-swine MHC class II DRα cDNA was inserted aft the second BiP IRES sequence in the Notl-Bglll site of pPBM4. pPB was designed to express the mini-swine MHC class II cDNA genes f DRα and DR3.

pPBM8: pPBM7 was modified to include a third BiP IRES sequen and the neomycin resistance gene. pPBM7 was designed to expres the mini-swine MHC class II cDNA genes for DRα and DR3, togeth with the neomycin resistance gene.

pPBM9: A 2.8 kb fragment was generated by PCR using the prime Hindlll-linked 5' DRjS (SEQ. ID. NO: 7) and ClaI-linked-3'SV40o ( SEQ. ID. NO: 5), utilizing pPBM8 as template. The fragment wa inserted into Hindlll and Clal digested pPBM4.

pPBMlO: pPBMlO is identical to pPBM9 except that the SV40 ori h been eliminated. A 2.62 kb fragment was generated by PCR usi primers Hindlll-linked 5' DRjS (SEQ. ID. NO: 7) and Clal-linked 3 Neomycin (SEQ. ID. NO: 19) and pPBMδ as a template. The D fragment was inserted into HindiII and Clal digested pPBM4.

Figure 2 shows a schematic diagram of the protocol used t construct pPBMl from pL7gCAT. In this case the Clal-SacI fragmen

of pL7gCAT was exchanged with that of pMPZen (Johnson et. al. 1989, EMBO J. , 8: 441-_448) in order to replace the MoMLV 3'LTR o pL7gCAT with a hybrid 3'LTR that contains the transcriptiona promoter enhancer region of Myeloproliferative Sarcoma Virus (M MPSV) . Jl, J2, J3 and J4 indicate the oligonucleotide primer used in polymerase chain reactions and correspond to SEQ. ID. NO 1, SEQ. ID. NO: 2, SEQ. ID. NO: 3 and SEQ. ID. NO: 4, respectively

Figure 3 shows an autoradiograph of a thin layer chromatograph plate depicting the results of an assay for the activity of th chloramphenicol acetyl transferase (CAT assay). In this assay Rous Sarcoma Viral promoter-driven expression of the CAT gen (pRSVcat - ATCC 37152) is the positive control for demonstratin the conversion of ¹⁴ C-labeled chloramphenicol to its acetylate monomeric form. All other vectors are as described in the text.

Figure 4 shows a bar graph demonstrating the amount of neomyci phospho-transferase polypeptide produced per ml of the COSM6 cel lysates following transient expression of the retroviral construct pPBM5. Neo-pl and Neo-flask refer to experiments demonstratin that pPBM5, containing the neomycin phosphotransferase gene, whe transfected into COSM6 cells grown on a tissue culture plate (Neo pl) or in a tissue culture flask (Neo-flask), respectively expressed high levels of neomycin phosphotransferase. RSV-p stands for a similar transfection with pRSV-CAT which was used a the negative control.

Figure 5 shows a flow cytometry analysis detailing the expressio of mini-swine MHC class II molecules following transien transfection of COSM6 cells.

A Contemporaneous histograms from a flow cytometry analysis o COSM6 cells transfected with pPBM4 which does not contain the DR sequence. No apparent shift in fluorescence intensity is observe

when the cells were stained with the MHC class II specif antibody, ISCR3, as compared to the fluorescence intensity of t cells stained with isotype control antibody 74-12-4.

B Contemporaneous histograms from COSM6 cells transfected wi pPBM7. The higher fluorescence intensity of staining of the cel with the ISCR3 antibody is indicated by the shift of the histogr obtained with ISCR3 as compared to that obtained with 74-12-4, t isotype control antibody.

Figure 6 is a flow chart summary for the experiments reported Example 2.

Figure 7 shows a flow cytometry analysis of the surface expressi of the mini-swine MHC class II DRα /β heterodimer from integrat polycistronic retroviral constructs.

The histograms were obtained by staining with the antibody 4 which specifically recognizes the DRα /β heterodimer and with 76-

11 antibody which is specific for IgG2b and which is the isoty control for 40D.

(A) and (B) - the staining patterns of untransfected GP/E-86 a

GP/E-envAM12 cells respectively.

(C) and (D) - the staining patterns of a mixed population ("bulk of G418 resistant sub-clones of GP/E-86 and GP/E-envAM respectively, that were transfected with pPBM8.

(E) and (F) - the staining patterns of cells that had be transduced with a mixture ("bulk") of sub-clones; (E) - packag virus from transfected GP/E-envAM12 cells were used to transdu the GP/E-86 cell line, described as GP>AM bulk; (F) - packag virus from transfected GP/E-86 cells had been used to transduce t

GP/E-envAM12 cell line, described as AM>GP bulk .

Figure 8 shows a flow cytometry analysis of individual G4

resistant sub-clones obtained after transfection with pPBM8. I each panel the histogram obtained by staining with 40D antibody wa superimposed on that obtained with 76-2-11. The cells were als stained with an MHC Class I antibody, B1094. (A) and (C) are th histograms obtained from untransfected GP/E-86 and GP/E-envAM1 cell lines, respectively. (B) is the histogram of the subclon GPl-2 which was derived by transfection of GP/E-86 cells wit pPBMδ. (D) is the histogram of the subclone Aml'.-ό which wa derived by transfection of GP/E-envAM12 cells with pPBMδ.

Figure 9 shows a photograph of an agarose gel showing the DN fragment produced by polymerase chain reaction using a prime specific for the 5' end of porcine DR/3 cDNA, Hindlll-linked 5' DR/ (SEQ. ID. NO. 7) in conjunction with a primer specific for the 3 end of porcine DRa cDNA, Bglll-linked 3'DRα ( SEQ. ID. NO. 16) The template DNAs used for the PCR analysis were obtained fro individual retroviral sub-clones, lane 1 is λ-BstEII DNA marker lane 2 is DFK cells, a negative control (Hirsch, F. et. al., 1992 J. Immunol. 149: 841-846 ) ; lane 3 is AMl^-δ; lane 4 is AMl ¹ _a -4 lane 5 is AMl' _c -3; lane 6 is GP2 ³ -1; lane 7 is GPθ'.-S; and lane 8 i GPl ² -2.

Figure 10 shows the Southern blot analysis of the chromosomal DNA from the transfected G418 resistant producer cell lines which wer generated using pPBMδ.

(A) shows an autoradiograph in which the respective names of th transfected cell lines are indicated at the top of each lane. Th radioactively labeled probe was a randomly primed porcine DR/3 cDN fragment. The sizes of the DNA fragments were estimated b comparing the mobilities of the bands to those of λBstEII DN markers. The estimated size of the DNA fragments in each lane i indicated by the arrow.

(B) is a schematic representation of the retroviral insert i pPBMδ. Sizes of DNA fragments which might have been generate

through potential deletional events occurring as a consequence o recombination between the BiP sequences are indicated.

Figure 11 shows a graphical illustration for each of vectors pPBM1 and pPBM14. pPBM13 is designed to express the DR/3 sequence unde the translational control of the 5'LTR and the DRα sequence unde the translational control of the BiP IRES sequence. pPBM14 i identical to pPBM13 except for the inclusion of the neomyci resistance gene which is under the control of the EMCV IRES.

Figure 12 shows a flow cytometry analysis of the expression of th mini-swine MHC class II DRα /β heterodimer on the surface of COSM cells transiently transfected with pPBM13. The histogram wa obtained by staining the cells with antibody 76-2-11 (an isotyp control), antibody 362 (specifically recognizes monkey class I) o antibody 40D (specific for MHC class II DRα /β heterodimer).

Figure 13 shows flow cytometry analyses of the expression of th mini-swine MHC class II ORa/β heterodimer on the surface of GP/E-8 cells. (A) GP/E-86 cells which had been transfected with pPBM which does not contain the sequences enabling ORa/β heterodime expression. (B) - (D) show histograms for individual G418 resistan clones, generated by transfection of GP/E-86 cells with pPBM14.

Figure 14 shows flow cytometry analyses of sub-clones derived fro PA317 cell line transduced with a transiently expressed vira supernatant from GP/E-66 cells, transfected with pPBM14. (A) is flow cytometry analysis of PA317 cells transduced with th supernatant obtained from mock-transfected GP/E-66 cells. (B)-(F are flow cytometry analyses for individual transduced G41 resistant sub-clones.

Figure 15 shows a bar graph detailing the percentage of cell expressing the MHC class II ORa/β heterodimer on the surface at an

given time, relative to the isotype control. The name of ea subclone is as indicated on the abscissa of the bar graph.

Figure 16 shows the results from slot blot hybridization experime of RNA isolated from the supernatants of G418 resistant sub-clone (A) Blot hybridized with randomly primed radioactively labeled D probe. (B) Blot hybridized with randomly primed radioactivel labeled DR/3 probe. The names of the sub-clones are as indicate

In one aspect, the invention provides retroviral vecto comprising (i) a single transcription regulatory sequence at the region thereof; (ii) a first DNA coding sequence; (iii) at lea one additional DNA coding sequence; and (iv) an IRES sequen controlling the translation of each such additional DNA codi sequence of (iii), wherein the vector expresses multip independent polypeptides capable of posttranslational combinati to form at least one functional multi eric protein. The retrovir vectors produce, for example, a functional heterodimeric protein the major histocompatibility complex.

In a preferred embodiment of this aspect, the IRES is a B IRES. Such vectors where the IRES is a BiP IRES preferably produ a single heterodimeric protein of the major histocompatibili complex. Exemplary vectors are selected from the group of pL7gCA pPBMl through pPBM14 and derivatives of any of them ("derivativ refers to structurally modified but substantially functional equivalent constructs). Also contemplated are cells containing t retroviral vectors of this aspect, which cells are preferab mammalian (particularly human).

Another aspect of the invention provides a retroviral vect comprising (i) a single transcription regulatory sequence at the region thereof; (ii) a first DNA coding sequence; (iii) at lea two additional DNA coding sequences; and (iv) IRESs controlling t

translation of each such additional coding sequence to express a least one functional multimeric protein, wherein at least one o such IRESs is a cellular IRES. Preferably, at least one suc cellular IRES is a mammalian IRES (e.g., a BiP IRES) and the othe IRESs can be viral IRESs, (e.g., encephalomyocarditis IRES, a poli virus IRES or a hepatitis virus IRES).

In another aspect, the invention provides a cell or tissu made to contain the retrovirus of the invention. The cell i preferably human.

In another aspect, the invention provides a method fo inducing immune tolerance in a human recipient for graf transplantation which comprises reintroducing, into said huma recipient, cells explanted from said human and made to contain vector of the invention. Preferably the DNA coding sequences cod for polypeptides of a functional heterodimeric protein of the majo histocompatibility complex of a non-human. Preferably the non human is porcine (e.g., miniature swine) or primate.

The protein product of the vector(s) of the invention is functional protein formed of multiple polypeptide subunits that ar combined by bridging moieties, or "linkers", e.g., disulfid bridges.

In one embodiment, for inducing donor specific tolerance in transplant recipient candidate, the vectors of the invention ar constructed to express both chains of at least one heterodimeri major histocompatibility complex protein.

In a particularly preferred embodiment of the presen invention, a polycistronic retroviral vector has been constructe which utilizes one copy of the 210 base pair 5' untranslate sequences from Human Immunoglobin Heavy Chain Binding Protei

(BiP). This expresses three polypeptides from a single transcrip all utilizing the BiP sequence. Also, one more expressible ge can be put under direct control of a 5' LTR and yet another und control of the SV40 promoter-enhancer.

In a particularly preferred embodiment of the prese invention, a polycistronic retroviral vector has been construct which utilizes one copy of the 210 base pair 5' untranslat sequence from Human Immunoglobin Heavy Chain Binding Protein (Bi and one copy of the encephalomycarditis IRES. A third polypepti is expressed under the translational control of the retroviral LTR. This vector expresses three polypeptides from a sing transcript, two of which post-translationally combine to form ultimeric protein of the major histocompatibility complex. Als one more expressible gene can be put under direct control of t SV40 promoter-enhancer.

The major advantages in using the BiP sequence are (i) i lack of secondary structure; (ii) absence of any internal A sequences; (iii) small size for viral packaging purposes; and (i most of all the ability to direct translational initiation in fr all three positions irrespective of the downstream distance fr the 5'LTR.

An additional novel aspect of this retroviral vector is t use of a hybrid 3' LTR in which the U5 sequence was taken fro Murine Proliferative Sarcoma Virus (M-MPSV) , and the R and sequences were taken from M-MLV, because it had been sho previously that M-MPSV bears a very strong transcriptional enhance which drives over-expression in yeloid tissues. (Bowtell, D.D. et. al., 1988. J.Virol. 62: 2464-2473) .

Examples of retroviral vectors which may be used inclu vectors derived from Moloney Murine Leukemia Virus, Spleen Necrosi

virus, Rous Sarcoma Virus and Harvey Sarcoma Virus. Specif vectors which may be constructed in accordance with the prese invention are described in the Examples.

EXAMPLE 1 CONSTRUCTION AND ANALYSIS OF POLYCISTRONIC RETROVIRAL VECTORS

Construction of pL7gCAT

The retroviral vector LNCX described in Miller and Rosma 1989, Biotechniques, 7: 960-990 was modified by eliminating t neomycin phosphotransferase gene and the CMV promoter a converting it to the polycistronic vector, pL7gCAT.

Construction of pPBMl

In this construction (Figure 1) the goal was to replace M-M 3'LTR from pL7gCAT with the 3'LTR from Murine Myeloproliferati Sarcoma virus (M-MPSV) which bears a relatively strong enhance promoter for transcription in cells derived from myeloid tissu This manipulation was done at the 3'LTR region because the 3'LTR duplicated during the retroviral integration process and there functions as the 5'LTR transcriptional enhancer-promoter from t integrated proviral state. A PCR fragment (Mullis, K.B. & Faloon F.A., 19δ7, Methods Enzymol., 155: 335-350; Saiki, R.K. et.al. 19δδ, Science: 239: 467-491) was generated using t oligonucleotide primers, termed J1(SEQ. ID. NO: 1) and J2(SEQ. I NO: 2) and the plasmid pMPZEN ( Johnson et. al., 1969, EMBO J., 6 441-446 ) . The primer Jl corresponds to nucleotides 2102 throu 2119 of M-MPSV (Genbank accession number K01683). The primer corresponds to the reverse complement of nucleotides 2693 throu 2709 of the M-MPSV sequence. The 608 base pair (bp) PCR fragme was digested with restriction endonucleases Clal and Sad electrophoresed through an agarose gel; the 551 bp Clal/SacI D fragment was excised and isolated using the Geneclean II D purification kit (Bio 101 Inc., La Jolla, CA) . pL7gCAT was als

digested with Clal and Sad, electrophoresed through an agaro gel. The PCR fragment that had been purified with the Geneclean solution was ligated to Clal/SacI digested pL7gCAT. The ligati mixture was used to transform E. coli JM109 (ATCC 53323). Positi sub-clones were identified by colony hybridization using a 32 labeled 104 bp PCR generated fragment using pMPZEN and J3 (SEQ. I NO: 3) and J4 (SEQ. ID. NO: 4) as the PCR primers. J3 correspon to nucleotides 2127 - 2143 and J4 to the reverse complement nucleotides 2215 - 2231 of M-MPSV. The clone pPBMl was subject to DNA sequence analysis, using Jl as primer to confirm t presence of the M-MPSV sequence in pPBMl.

Analysis of CAT expression from pL7gCAT and pPBMl

COSM6 cells (a transformed African Green Monkey Kidney ce line) were transfected (Aruffo, A. and Seed, B., 1987, Proc. Nat Acad. Sci. U.S.A. 84: 8573- 8577) with pPBMl and pL7gCAT and test for CAT expression (Gorman et. al., 1982,. Mol. Cell Biol. 2: 1044 1051; Prost. E. and Moore, D., 1986, Gene 45: 107-111 ). C activity was not detectable from transfections using either of t two plasmids. There were two possible explanations for the lack CAT expression:- (1) that the preliminary DNA sequence analysis pL7gCAT was incorrect and (2) that, since neither vector contain the SV40 ori, the lack of gene expression was a consequence of t inability of the vectors to undergo DNA replication in COSM6 cells DNA sequence analysis of pL7gCAT and pPBMl revealed that, while t initiator methionine and the following sequence were intact in t CAT gene, there was a deletion of 11 nucleotides between the 3 junction of the third BiP sequence and the CAT gene. In order establish whether or not those 11 nucleotides were essential f internal translational initiation, the SV40 ori sequence w inserted into pPBMl. This vector is described as pPBM2.

Construction of pPBM2.

A PCR fragment was generated using ClaI-linked-3'SV40ori (SE

ID. NO: 5) and ClaI-BglII-linked-5'SV40ori (SEQ. ID. NO: 6) primer with pCDM8 plasmid DNA (Seed, B., 1987, Nature 329: 840-842) as th template. The PCR fragment was cleaved by Clal and gel purified The Clal-digested PCR fragment was subcloned in the Clal site o pPBMl. In this construct, the SV40ori sequence went in th orientation such that the Bglll site is located adjacent to th 3'LTR (See Figure 1: pPBM2 ).

Analysis of CAT expression from pPBM2

COSM6 cells were transfected with pPBM2 and analyzed for th production of CAT. The transient expression of CAT harvested fro COSM6 cells transfected with pPBM2 was comparable to the level o CAT produced by cells transfected with the control plasmid pRSVCA (Figure 3). These results indicate that the deletion of the 1 nucleotides from pL7gCAT did not result in the lack of apparen expression of the CAT gene in vectors pL7gCAT and pPBMl. Rather the lack of expression was a consequence of the inability of thes vectors to replicate in COSM6 cells.

Construction of pPBM3: The next vector in the series of vector which would ultimately result in a vector capable of expressing th mini-swine MHC class II ORa/β cDNA genes was one which containe the mini-swine MHC class II DR/3 cDNA gene. The DR/3 cDNA-containin plasmid pPBSKSII-DR/3 (Gustafsson, K. et. al. , 1990, Proc. Natl Acad. Sci. U.S.A. 87: 9798-9602; Shafer, G.E. et. al., 1991, Proc Natl. Acad. Sci. U.S.A. 88: 9760-9764) was used as a template, i a PCR reaction in the presence of primers Hindlll-linked 5'DR/ (SEQ. ID. NO: 7) and Notl-linked 3' DR/3 (SEQ. ID. NO: 8). Th fragment was cleaved with HindiII and NotI and inserted between th Hindlll and NotI sites of pPBM2. The vector generated is describe as pPBM3. Analysis of the transient expression of the CAT gene fro pPBM3 (Figure 3) indicated that the insertion of the DR/3 cDN sequence did not affect the level of CAT expression, relative t pPBM2.

Construction of pPBM4 and pPBM4-prime

A polylinker sequence containing BstXI, Sspl and Ml restriction sites was created by annealing two 25 bp lo complementary oligonucleotides polyl (SEQ. ID. NO: 9) and pol (SEQ. ID. NO: 10) and inserting the fragment into the EcoRI site pPBM3, thereby generating pPBM4. DNA sequence analysis of pPB revealed that three copies of the polylinker were present in t plasmid. For subsequent constructions it was necessary to chan the orientation of the SV40 ori such that the Bglll site would located at the 5' end of the SV40 ori fragment. Hence, the SV40o fragment was excised and re-subcloned in Clal digested pPB vector, generating pPBM4-prime. During the construction of pPBM4 prime the polylinker was eliminated, . the EcoRI site was re-create and the Clal site at the 3' end of SV40ori sequence was destroye

Construction of pPBM5: A BiP IRES-containing DNA fragment wa generated from pL7gCAT by PCR using the primer Notl-linked 5 primer(SEQ. ID. NO: 11), which contains the nucleotides 376 - 40 of the human GRP78 sequence (Genbank Accession number M19645) an a NotI site, and primer EcIR (SEQ. ID. NO: 12), which contains th reverse complement to nucleotides 569-587 of the human GRP7 sequence (Genbank Accession number M19645) and an EcoRI site. Th PCR fragment was cleaved with NotI and EcoRI and purified by ge electrophoresis and GeneCleanll. A neomycin phosphotransferas resistance gene-containing DNA fragment was generated by PCR usin primer EXneo(SEQ. ID. NO: 13), which contains nucleotides 149 - 17 of the neomycin resistance gene (Genbank Accession number J01834) and sites for EcoRI and Xhol, and primer Bglll-linked 3' neomyci (SEQ. ID. NO: 14), which contains the reverse complement to th neomycin resistance gene nucleotides 922 - 945 and a Bglll site The plasmid pSV2Neo (ATCC 37149) was used as the template in th PCR reaction. The PCR fragment was cleaved with EcoRI and Bglll an purified by gel electrophoresis and GeneCleanll .The two PC fragments were directionally inserted in NotI and Bglll digeste

pPBM4-prime, giving rise to pPBM5 (Figure 1).

Analysis of neomycin phosphotransferase activity from pPBM5: COS were transfected with pPBM5. 46 hr after transfection the cell were harvested and analyzed for the level of neomyci phosphotransferase activity expressed intracellularly using a NPTI ELISA kit #5307-543210(Five-Prime To Three-Prime, Boulder, CO) The results of the assay are presented in Figure 4.

Construction of pPBM7: pPBM4 prime was used as the vector fo subcloning the porcine DRα gene. Porcine MHC class II DRα gen (Hirsch et al., 1992, J. Immunol. 149, 841-646) was derived usin Sail-linked 5' primer(SEQ. ID. ^; N0: 15) and Bglll-linked 3 primer(SEQ. ID. NO: 16) using pPBSKSII-DRα ^c as template plasmid The BiP sequence was isolated by PCR from pL7gCAT using Notl-linke 5' primer (SEQ. ID. NO: 11) and Sail-linked 3' primer (SEQ. ID. NO 17). The vector was generated by digesting pPBM4 -prime with Not and Bglll. The BiP IRES sequence and DRα sequence were the directionally subcloned in pPBM4 - prime, to give rise to pPBM7 pPBM7 contains the sequences for the mini-swine MHC class II cDN genes for DRα and DR/3.

Construction of pPBM8: pPBM7 was digested with Bglll and the 5' termini of the vector were dephosphorylated, using calf intestina alkaline phosphatase (Promega Corp. Madison, I). The BiP-Neomyci fragment to be inserted at the Bglll site was isolated by PCR usin Bglll-linked 5' BiP primer (SEQ. ID. NO: 18) and Bglll-linked 3 Neomycin primer (SEQ. ID. NO: 14) using pPBM5 as the template. T determine the orientation of the fragment in the sub-clones, DN from the sub-clones was analyzed by digestion with a combination o several restriction enzymes. pPBMδ contains the sequences for th mini-swine MHC class II cDNA genes for DRα and DR/3 together wit the G416 resistance gene.

Construction of pPBM9: After finishing the construction of pPBMδ it was discovered that the polylinker sequence had been eliminat at the time of constructing pPBM4-prime from pPBM4. Therefore, order to insert the polylinker sequence into pPBMδ, a 2.6 kb P fragment was generated using pPBMδ as template and the prime Hindlll-linked 5' DR/3 (SEQ. ID. NO: 7) and Clal-linked 3'SV40o (SEQ. ID. NO: 5). The PCR fragment was cleaved with Hindlll a Clal and inserted between the Clal and NotI sites of pPBM4, there creating pPBM9. pPBM9 contains the sequences for the mini-swi MHC class II cDNA genes for DRα and DR/3.

Construction of pPBMlO: A PCR fragment was generated using prime Hindlll-linked 5' DR/3 (SEQ. ID. NO: 7) and Clal-linked 3' Neomyc (SEQ. ID. NO: 19) and pPBMδ as the template. The PCR fragment w cleaved with Hindlll and Clal and purified by gel electrophores and GeneCleanll. The fragment was inserted into the vect fragment of NotI and Clal digested pPBM4. , thereby generati pPBMlO. pPBMlO contains the sequences for the mini-swine MHC clas II cDNA genes for DRα and DR/3 but differs from pPBM9 insofar as t SV40ori sequence was eliminated. pPBMlO was used for t generation of the retroviral packaging cell lines, designed express the mini-swine MHC class II ORa/β heterodimer.

Expression of mini-swine MHC class II DRα//3 from the polycistron retroviral vectors. COSM6 cells were transiently transfected wi (A) pPBM5 or (B) pPBM7. After 48 hr the cells were analyzed f the cell surface expression of the MHC class II ΩRa/β heterodime For detection of class II gene expression on the surface of t cells, the primary antibody used was ISCR3, which specificall recognizes an epitope of the DRα only when both DRα and DR/3 a presented as a heterodimer on the surface of the cell ( atanabe, et al., 1983, Transplantation 36: 712 -718). As a control for t staining procedure, the cells were also stained with the W63 antibody (Pescovitz, M.D. et al., 1984, J. Exp. Med. 160: 1495

1505) that specifically recognizes monkey MHC Class I protein. T primary antibody reaction was followed by incubating the cells wi the secondary antibody, fluorescein isothiocyanate (FITC) conjugated goat anti-mouse immunoglobulin. The stained cells we analyzed using a FACScan flow cytometer (Becton Dickinson, S Jose, CA) . Figure 5 shows the data displayed in a histogram form as cell number (ordinate) vs. logarithm of fluorescence (abscissa) Dead cells were eliminated from the analysis by appropriate gatin In every instance, the data was matched and compared with the da obtained using a corresponding isotype control for that particul antibody. (76-2-11 for ISCR3 and 74-12-4 for W362). The superimpos histograms (Figure 5) show that 10-15% of the COSM6 cel transfected with pPBM7 which contains both the DRα and DR/3 cD genes in a polycistronic fashion manifested enhanced fluorescen intensity over cells transfected with pPBM5, which expresses on the DR/3 cDNA gene. The present data, therefore, emphasize that o polycistronic retroviral vectors are capable of expressing t heterodimeric ORa/β on the cell surface.

This demonstrates a system in accordance with the inventi whereby expression of multiple genes in recombinant retroviral DN based constructs can readily be tested in transiently transfect C0SM6 cells without doing a stable selection or without the ne for packaging the recombinant retroviral DNA within its co protein.

EXAMPLE 2 GENERATION OF RETROVIRAL PRODUCER CELL LINES EXPRESSING

PORCINE MHC CLASS II ORa/β HETERODIMER USING pPBMδ This example reports the packaging of the constructs, capab of expressing three genes in a polycistronic fashion, with retroviral coat proteins and their ability to transduce cell lin A flow-diagram of the experiments reported in this Example is sho in Figure 6. High level expression was monitored from all thr

genes from the G418 resistant clones, thereby conclusivel demonstrating successful utilization of the BiP IRES sequence in polycistronic fashion.

Transfection & Transduction of packaging cell lines:

The pPBM8 retroviral vectors were used to transfect, using transfection kit (Stratagene, CA) , either the ecotropic packagin cell line, GP/E-86 or the amphotropic packaging cell line GP/E envAM12., both packaging cell lines were obtained from Dr. Arthu Bank (Columbia University). Forty-eight hours after transfection transiently expressed virus in the supernatant was recovered b filtration through a low protein binding syringe filter and adde to a 20% to 30% confluent plate of recipient cells for transductio in presence of 8 mg/ml Polybrene (Sigma, St Louis, MO). GP/E envAM12 cells were the recipient cells for the supernatant fro GP/E-86 cells and vice versa. After 4 hours of infection, th viral supernatant was removed and complete medium was added t allow the cells to undergo 2-3 cycles of replication in 2-3 days At this point the transduced cells were split in several dilution and 0.6 mg/ml of active G416-containing medium was added. Unde these growth conditions, only the cells expressing the neomyci phosphotransferase gene survived . After 14 days of selection i G41δ-containing medium, transduced sub-clones were picked an expanded, either as isolated sub-clones or mixtures of sub-clones Meantime, the transfected cells were also grown in selective mediu and subsequently analyzed for ORa/β expression.

Analysis of populations of cells transduced by recombinan retrovirus containing mini-swine MHC class II DRa/b cDNA genes:

Flow cytometry analyses: Flow cytometry analyses of the transduce cell lines were performed with antibodies such as ISCR3 ( atanab et al. supra) or 40D (Pierres, M. et al., 1980, Eur. J. Immunol 10: 950 -957), both of which recognize porcine MHC classII-DR polypeptide only when it forms a heterodimer with the DR _/

polypeptide. Figure 7 shows the data obtained from the mixture o sub-clones, referred to as "bulk". Neither of the untransfecte cell lines exhibit enhanced fluorescence staining with the anti class II antibody 40D (see Figure 7A and 7B). The mixture of sub clones transfected with pPBMδ show a slight shift (see Figure 7 and 7D) while the transduced cells show a clear shift o fluorescence intensity (see Figure 7E and 7F). Indicative of th fact that the virus made by GP/E-envAM12 cells have transduced th GP/E-66 cells more efficiently so as to give rise to bette expression of the ORa/β heterodimer is evident by the remarkabl shift obtained by staining with 40D (Figure 7F) relative to tha with the corresponding isotype. A statistical analysis indicate that 35% of the transduced G418 resistant GP/E-86 (denoted a AM>GP) cells were capable of expressing the ORa/β heterodimer o the surface.

Analysis of individual sub-clones generated by transfection o packaging cell lines using pPBMδ:

After analyzing the bulk population of transfected an transduced cells by flow cytometry, the bulk population was score for individual G418 resistant sub-clones by limiting dilution Individual sub-clones were subsequently analyzed by flow cytometry The representative fluorescence histograms for an ecotropic as wel as an amphotropic producer subclone are shown in Figure 8. Figur 8A shows the superimposed histograms of the control (untransfected G418 sensitive) GP/E-86 cells, obtained by staining with surfac antibody 40D for porcine ORa/β heterodimer and with the isotyp control antibody 76-2-11. The histogram obtained by staining cell with the antibody B1094 (Dr. D. H. Sachs, Massachusetts Genera Hospital, Boston, MA), specific for mouse MHC Class I antige served as a positive control for successful staining. Absence o any shift of peak of the fluorescence intensity in the 40D hiεtogram with respect to that of the isotype histogram indicate that the untransfected packaging cell line does not express the MH

class II ORa/β heterodimer (Figure 8A) . However, as shown i Figure 8B, the transfected cell line GP12-2 gives rise to histogram which shows a clear shift of a fluorescence peak with th 40D antibody. The untransfected cell line GP/E-envAM12 does no express the MHC class II ORa/β heterodimer (Figure δC), while th transfected cell line AMl'.-δ shows a high level of expression o the ORa/β heterodimer (Figure 8D) . Statistical analyses a obtained by legitimate gating of the fluorescence histograms showe that 42% and 65% of the total cell population express ORa/ heterodimer from the ecotropic and amphotropic producer sub-clone respectively.

Analysis of the integrated DNA

Using cellular DNA as the template, polymerase chain reaction were performed using primers Hindlll-linked 5'DR _/ 3 (SEQ. ID. NO: 7 and Bglll-linked 3'DRα (SEQ. ID. NO: 16) to analyze the integrate DNA fragments present in the chromosomes of the individual produce sub-clones. PCR fragments were sequenced in order to ascertain th integrity of the DNA, i.e., to ensure that the recombinan retroviral vector has integrated in the cellular chromosome withou undergoing any gene rearrangement within the vector sequence

Appearance of an 1.8 kb PCR fragment was as expected from th theoretical estimation (Figure 9). The sequence analysis reveale that the integrated genome did not undergo any rearrangement durin the first week of cellular expansion. The generation of a shorte band which appeared even in the negative control was either a artifact or had been generated from the non-specific annealing o the primers at some cellular sequences.

Viral titer

The titer of virus stocks produced from both the producer cel lines on NIH-3T3 cells were determined to be about 2 x 10 ⁵ .

Thus, these data strongly support the effectiveness of usin

a polycistronic vector in expressing multimeric protein for t purpose of retroviral gene therapy.

EXAMPLE 3 ANALYSIS OF DNA FROM CELLS TRANSFECTED WITH pPBMδ During continued passage of the producer cell lines obtain after transfecting with pPBMδ, it was noted that the cel gradually lost the ability to express the mini-swine MHC class ORa/β heterodimer. Southern blot analysis of the integrated D was performed. Genomic DNA was isolated from the cell lines rinsing a 100 % confluent culture plate twice with IX phospha buffered saline (PBS). The cell were lysed by the addition 400 μl 2X lysis buffer (2X lysis buffer contains 0.2 M Tris-HCl, 7.0, 0.1 M Na ₂ EDTA, 2% SDS, 0.1 M NaCl, 400 μg/ml proteinase K. T lysed cells were scraped at room temperature and incubat overnight at 55oC. The solution was then extracted five times wi buffer-saturated phenol-chloroform. The DNA was ethano precipitated and re-dissolved in 10 mM Tris-HCl, pH 7.0 , 1 Na2EDTA, 50 μg/ml DNase-free RNase at 37°C for 1 hour. The DNA w re-extracted with phenol-chloroform and ethanol-precipitated. T DNA was re-suspended in 10 mM Tris-HCl, pH 7.0, 1 mM Na ₂ EDTA. F Southern blot analysis the DNA (20 μg) was digested with Sad whi cleaves the retroviral vectors only in the 5' and 3' LTRs.. T digested DNA was electrophoresed through 1% agarose and transferr to a nitrocellulose filter (Scheicher and Schuell, Keene, NH) . T membrane was hybridized overnight under the appropriate conditio for a randomly primed radioactive (using the method described the Boehringer-Mannheim random priming kit) probe (either the D or the DR/3 fragment) . The membranes were washed under stringe conditions and exposed to Kodak X-OMAT film. Figure 10A shows autoradiograph of Sad digested DNA from the cell lines probed f the presence of sequences hybridizing to the pig DR/3 probe. T multiplicity of bands corresponding to 4.9 kb, 3.6 kb and 3.0 can be interpreted using the schematic diagram in Figure 10B a

correspond to the sizes expected if the integrated DNAs had undergone homologous recombination between the copies of the BiP IRES sequences. The frequency of the deletion events in the transfected retroviral packaging cell lines can be explained by the ability of the produced virus to super-infect the host cell and subsequent retrovirus-mediated homologous recombination. The phenomenon of super-infection has been reported (Muenchau et. al., 1990, Virology 176: 262-265). There are several recent reports in the literature regarding the occurrence of retrovirus-mediated homologous recombination (Zhang and Temin, 1993, Science, 259: 234- 238; Temin, H., 1993, Proc. Natl. Acad. Sci. USA, 90: 6900-6903). The presence of bands corresponding to sizes that cannot be readily explained may be the result of recombination events between short stretches of non-obvious sequence homology. It is of interest to note that the intensity of the band corresponding to the full- length proviral DNA sequence (the 4.8 kb band) correlates, for different sub-clones, with the level of expression of the ORa/β heterodimer, (Figure 8)

EXAMPLE 4

GENERATION AND ANALYSIS OF PACKAGING CELL LINES RETROVIRALLY

TRANSDUCED WITH PPBM14 Construction of pPBM13 pPBM7 (Figure 1) was modified so that the DR/3 cDNA gene could be under the direct control of the 5'LTR for translational initiation. In order to do this, the EcoRI-BspEI fragment which contains the 5' cDNA sequence of DR/3 from pBSKSII-DR/3 plasmid (Gustaffson, K. et al., 1990, Proc. Natl. Acad. Sci. USA, 87: 9798- 9802) was used to replace the similar fragment from pPBM7 which contained not only the 5' sequence of DR/3 but also the first BiP IRES sequence. The new construct, pPBM13, (Figure 11), thus contains the DR/3 gene under the translational control of the 5'LTR. The DRα cDNA gene remains under the translational control of the

BiP IRES. pPBM13 does not contain sequences for selection eukaryotic cells.

Construction of pPBM14

In order to incorporate a drug resistance marker, pPBM13 w modified as follows. A DNA fragment containing a second, no homologous IRES, was generated using the polymerase chain reacti - the plasmid pGlEN (Genetic Therapy Inc. Gaithersburg, MD) w subjected to the polymerase chain reaction in the presence primers Bglll-XhoI-linked 5' EMC (SEQ. ID. NO: 20) and Bglll-link 3' neomycin (SEQ. ID. NO: 14). The fragment thus amplifi contains the 5' untranslated region of the encephalomycardit virus (EMCV) and the neomycin phosphotransferase gene. The D fragment was cleaved with Bglll and gel purified before bei inserted into the Bglll site in pPBM13, thereby generating pPBM (Figure 11) .

Analysis of MHC class II DRa/b expression following transfecti of pPBM13 into COSM6 cells:

Transfection into COSM6 .cells was performed, using Cs purified supercoiled plasmid DNA, pPBM13, by the DEAE dextr sulfate method as described in Example 1. 48 hr followi transfection, the cells were harvested by scraping the cultu plates in the presence of IX phosphate-buffered saline (PBS - PBS is 137 mM NaCl, 2.7 mM KC1, 4.3 mM Na ₂ HP04.7H ₂ 0, 1.4 mM KH ₂ P0 pH 7.3) containing 0.2% Na ₂ EDTA. The collected cells were rins twice with IX PBS and 10 ⁶ cells (in 100 μL) were added to each we of a 96 well micro-titer plate. Appropriately diluted prima antibodies were added to the cells which were stained for 30 mi at room temperature. The non-reactive antibodies were washed o and FITC-conjugated secondary antibody was added for 30 min. 4°C. After stringent washing of the cells, the cells were analyz by flow cytometry. Phosphor-iodide gating (Becton-Dickins Manual) was used to gate out dead cells. Figure 12 shows t

results of the flow cytometry analysis, the data is represented i histogram format using the number of cells as the ordinate and th fluorescence intensity as the abscissa. It is apparent from Figur 12 that the vector pPBM13 expresses the mini-swine MHC class I ORa/β heterodimer as efficiently as did pPBM7.

Transfection, using pPBM14, into GP/E-86 and subsequen transduction into the PA317 retroviral packaging cell line.

The ecotropic GP/E-86 retroviral packaging cell line wa transfected with pPBM14 using the Mammalian Transfection Ki (Stratagene, La Jolla, CA) The transiently expressed virus fro GP/E-86 cells was harvested 46 hr after transfection, filtere through a low protein binding 0.45 μM filter (Gel an Sciences, An Arbor, MI) and was used to transduce the amphotropic packaging cel line PA317 (ATCC CRL 9078). After transducing the cells for lδ h with a retroviral titer of 104/ml, the transduced cells were spli in appropriate dilutions and 500 μg/ml active G416 was added to th medium in order to select G418 resistant cells. To monitor th transfection efficiency, the transfected GP/E-86 cells were plate in presence of 1 mg/ml active G418 containing media in limite dilutions for scoring the G418 resistant clones.

Analysis of MHC class II ORa/β expression following transfection o pPBM14 into (a) GP/E-86 cells and (b) subsequent transduction o PA317 cells

The results of flow cytometry analysis of GP/E-86 cells stabl transfected with pPBM14 are shown in Figure 13. All of the G41 resistant cells expressed the MHC class II ORa/β heterodimer, t differing extents (see Figure 13 for the flow cytometry analysis o the four individual transfected G418 resistant sub-clones).

Transiently expressed ecotropic retrovirus harvested 46 hr after transfection into GP/E-86 cells was used to transduce th amphotropic cell line PA317 Transduced sub-clones were obtaine

following selection in G418. Flow cytometry analysis of the G4 resistant clones revealed that more than 90% of the individu transduced sub-clones were positive for expression of the MHC cla II ORa/β heterodimer. Figure 14A shows the negative control, i. PA317 cells transduced with supernatant generated from moc transfected GP/E-86 cells. The majority of the histograms obtain with antibody 40D showed a single clear shift of peak fluorescen intensity (five representative histograms are shown in Figure 14 F). One histogram (Figure 14F) showed a double shift of pe fluorescence intensity, probably indicating a mixed population MHC class II ORa/β heterodimer expressing sub-clones.

Figure 15 displays a bar graph showing the percentage transduced cells in a particular population which demonstrate higher level of fluorescence intensity with the antibo recognizing the ORa/β heterodimer relative to that recognizing t isotype control.

In order to determine whether the producer clones lose the ability to express the MHC class II ORa/β heterodimer, sever clones were expanded in the presence of sub-optimal concentratio of G418 (200 μg/ml for the PA317-derived lines and 500 μg/ml f the GP/E-66 derived lines. After approximately 30 cell divisio the producer clones were re-analyzed by flow cytometry analysi The expression levels remained constant, indicating that t integrated retroviral sequences were not undergoing the rap rearrangement/deletion phenomenon that had been observed with t lines derived from pPBMδ (data not shown) .

Analysis of viral RNA

In order to ascertain that the expression of the MHC class ORa/β heterodimer was the consequence of expression of sequenc present in a single proviral integration event, RNA was extract from pelleted virus and an RNA slot blot analysis was performe

The autoradiograph in Figure 16 indicates that there are equivalen amounts of DRα and DR/3 RNA for the clones as discerned by th intensity of hybridization with the respective cDNAs as radioactiv probes. This argues in favor of the integrity of the provira genome in the producer sub-clones. Had recombination/deletio occurred we would have expected a difference in the proportionalit of the RNA transcripts specific for either DRα or DR/3 because it i likely that viruses bearing two different types of RNA genome would express different amounts of RNA.

Assay for viral titer.

Virus was harvested from virus producing cells by adding 5 m of fresh culture media on day 1 to 60% confluent 10 cm dishes fo 16 hr. On day 2, the supernatant containing the virus was filtere through 0.45 μM low protein binding filter and dilute appropriately with media. Then, 0.5 ml of the supernatant fro various dilutions was added to 40% confluent dishes of recipien NIH-3T3 (ATCC CRL 1656) cells in presence of 4 μg/ml of Polybrene On day 3, fresh media containing 1 mg/ml active G418 was added t the transduced cells and neomycin resistant colonies were score after 10-12 days by fixing the cells in ethanol and staining th cells with Giemsa stain. Virus titer was calculated in colon forming units/ml (cfu/ml) as shown in Table 1.

Assay for presence of replication competent retrovirus b amplification of transduced NIH/3T3 cells.

The stable G418 resistant NIH-3T3 cells were amplified an tested for presence of replication competent retrovirus (RCR) . I any contamination or recombinatorial event was able to give ris to RCR in the amphotropic viral supernatant that was used to infec NIH-3T3 cells, expansion of the NIH-3T3 cells would result in several-fold amplification of the RCR. By this amplificatio technique, breakout of a single RCR could be detected by doin marker rescue S+L- assay using PG4 as an indicator cell lin

(Haapala, D. et al., 1965, J. Virol. 53: 627-633 )or by PCR usin primer sequences corresponding to the sequences of envelop protein. Details of these methods are as described (Anderson, W.F. 1993, Human Gene Therapy, 4: 31-321). The inability of th supernatants from the respective NIH-3T3 cells to form foci on th PG4 cells indicated that replication competent virus was no present or, had not been generated in this particular combination of retroviral vectors and packaging cell lines.

SEOUENCE LISTING

(1) GENERAL INFORMATION:

(i ₎ APPLICANT(S): Banerjee, Papia T.

LeGuern, Christian A. Seed, Brian ₍ ii ₎ TITLE OF INVENTION: polycistronic Retroviral Vector Containing Multipl Related Translational Initiation Capability (iii) NUMBER OF SEQUENCES: 20 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Carella, Byrne, Bain,

Gilfillan, Cecchi, Stewart & Olstein

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(C) CLASSIFICATION: U.S. Preliminary Class 435 (viii) ATTORNEY/AGENT INFORMATION:

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(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 201-994-1700

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(2) INFORMATION FOR SEQ ID NO: 1 (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 17 BASES

SUBSTITUTE SHEET (RULE 26]

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 1: GCTCTTCGGA CCCTGCA 17

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(A) LENGTH: 17 BASES

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO. CGAGTGAGGG GTTGTGG 17

(4) INFORMATION FOR SEQ ID NO: 3 (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 17 BASES

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(C) STRANDEDNESS: SINGLE

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO. CGATTAGTCC AATTTGT 17

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(A) LENGTH: 17 BASES

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(C) STRANDEDNESS: SINGLE

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO. TCTATCTATG GCTCGTA 17

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(A) LENGTH: 27 BASES

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR (iii) HYPOTHETICAL: NO (iv) ANTISENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 5: GTCAATCGAT AGCTTTTTGC AAAAGCC 27

(7) INFORMATION FOR SEQ ID NO: 6 (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 33 BASES

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 6: GCGAATCGAT AGATCTACTC CGCCCATCCC GCC

(8) INFORMATION FOR SEQ ID NO: 7 (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 30 BASES

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(D) TOPOLOGY: LINEAR (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 7: GGCTCAAGCT TCAGCATGGC ATGTCTGTGT 30

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(A) LENGTH: 45 BASES

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR (iii) HYPOTHETICAL: NO (iv) ANTISENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 8: AAGGAAAAAA GCGGCCGCTT ATCAGGAGGC CTGTTGGCTG AAGGG 45

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(A) LENGTH: 25 BASES

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 9: AATTGGCCAA TATTTTGGAC GCGTC 25

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 10: ATTGACGCGT CCAAAATATT GGCC 24

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(D) TOPOLOGY: LINEAR (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 11:

AAGGAAAAAA GCGGCCGCCG CGACGCCGGC CAAGACAGCA CAGA 44

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(A) LENGTH: 28 BASES

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(D) TOPOLOGY: LI EAR (iii) HYPOTHETICAL: NO (iv) ANTISENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 12:

GATCGAATTC AGCCAGTTGG GCAGCAGC 28

(14) INFORMATION FOR SEQ ID NO: 13 (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 47 BASES

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOG : LINEAR (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO. 13:

TCGAATTCCA AGGCTCGAGC ATGATTGAAC AAGATGGATT GCACGCA 47

(15) INFORMATION FOR SEQ ID NO: 14 (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 36 BASES

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOG : LINEAR (iii) HYPOTHETICAL: NO (iv) ANTISENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 14:

CACAAGATCT TATCAGAAGA ACTCGTCAAG AAGGCG 36

(16) INFORMATION FOR SEQ ID NO: 15 (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 33 BASES

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO. 15:

TTCCGGTCGA CGCCGCCAAA ATGACCATAC TTG 33

(17) INFORMATION FOR SEQ ID NO: 16 (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 36 BASES

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR (iii) HYPOTHETICAL: NO (iv) ANTISENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 16:

GGACAGATCT TATCACAGAG GCCCTCGGTG TTCAGT 36

(18) INFORMATION FOR SEQ ID NO: 17 (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 36 BASES

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 17:

GATCAGCCGG TCGACAGCCA GTTGGGCAGC AGCAAG 36

(19) INFORMATION FOR SEQ ID NO: 18 (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 33 BASES

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR (iii) HYPOTHETICAL: NO (iv) ANTISENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 18:

GGAATCAGAT CTCGACGCCG GCCAAGACAG CAC 33

(20) INFORMATION FOR SEQ ID NO: 19 (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 33 BASES

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 19:

CCATCGATGG TAATCAGAAG AACTCGTCAA GAA 33

(21) INFORMATION FOR SEQ ID NO: 20 (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 32 BASES

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 20: CAGGAAGATC TCTCGAGGAT CAATTCCGCC CC 32

TABLE1

TΓTFR.CFU/ML.

3 x 10 ⁴ 1 xlO ³ 6xl0 ³ 5xl0 ⁴ 1 xlO ⁶ 1 xlO ⁶ 2xl0 ⁵ 1 xlO ⁶

4 x 10 ⁴ 1 xlO ⁵

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